Aerobic metabolism II: Electron transport and oxidative phosphorylation (main)

Provide an example that is similar to harvesting energy from NADH and FADH2 (SKIP for fast)

Provide an example that is similar to harvesting energy from NADH and FADH2 (SKIP for fast)

The hydro-electricity generator which spins with the movement of water which is just like the ATP-Synthase which spins after H+ ions enter through it down their steep electrochemical-gradient which provides energy to produce ATP from ADP and Pi

1. Why oxygen and not any other molecule

2. What allows it to recieve electrons

   1. disadvantage of it

1. Because:

   1. Oxygen is found everywhere

   2. Oxygen passes easily through membranes because it is small non-polar

   3. it accepts electrons

2. It’s Diradical structure (two unpaired electrons)

   1. The disadvantage of using oxygen is that the same property that allows for accepting electrons allows for formation of ROS which are toxic

Define :

1. Electron transport chain

   1. What is it’s main function

   2. What are the secondary functions

2. Aerobic respiration

1. Electron transport chain is a series of electron carriers arranged in the inner mitochondrial membrane in order of increasing electron affinity

   1. transfer electrons from NADH and FADH2 all the way to an O2 molecule while pumping H+ ions to mitochondrial matrix setting the stage for ATP synthesis

   2. Secondary functions:

      1. Transfer Ca2+ into the mitochondrial matrix via Mitochondrial associated membranes

      2. Generate heart in brown adipose tissue

2. Aerobic respiration is the coupling of electron transfer ultimately to ATP synthesis

1. What do the Complexes share in common

2. Label the parts of electron transport chain and their Other name

1. They all are:

   1. Oxidoreductases

   2. Consists of several proteins and prosthetic groups

   3. All located in the inner mitochondrial membrane

2. Complexes

   1. Complex I (NADH dehydrogenase)

   2. Complex II (Succinate dehydrogenase)

   3. Complex III (Cytochrome bc1 complex)

   4. Complex IV (cytochrome oxidase)

1. What moves electrons between the Complexes

   1. What is its oxidization states and their names

   2. structure note

   3. Polarity and where does it move

   4. from where to where

1. Ubiquinone:

   1. Oxidation states :

      1. UQ (oxidized form) (Ubiquinone)

      2. UQH (ubisemiquinone)

      3. UQH2 (reduced form) (dihydroubiquinone)

   2. Ubiquinone has repeating units of (isoprene) which can change from species to another but in mammals generally it is n=10

   3. it is hydrophobic so it move only through the inner membrane

   4. Transfers electrons from Complex I and Complex II (and two other enzymes sometimes) to complex III

2. Cytochrome C (Cyt C):

   1. Oxidation states :

      1. Oxidized form : Cyt Cox (Fe3+)

      2. Reduced form: Cyt Cred (Fe2+)

   2. Structure :

      1. It houses a heme group that can be oxidized and reduced with 1 electron each

   3. It is hydrophilic and moves through the intermembrane space

   4. It transferes electrons from Complex III to Complex IV

1. What can be the prosthetic groups of complexes

2. what are their functions

3. why do we have different ones

1. Types :

   1. FMN Carries 2 electrons at a time

   2. FAD Carries 2 electrons at a time

   3. FeS : Carries 1 electron at a time with Two forms :

      1. 2 Fe to 2 S where irons also bind to 2 S of cysteines

      2. 4 Fe to 4 S where iron only binds to 1 S of cystine each

      3. Not that an Fe should bind to 4 S and a sulfur should bind to two other stuff (Fe or cysteine)

   4. Heme Carries 1 at a time

   5. Copper Carries 1 at a time

2. They can be oxidized and reduced to carry electrons from one part to another

3. we have different ones because each one can have different affinity to electrons to be able to take from the other easily

What prosthetic groups do each Complex have

1. Complex I: FeS , FMN

2. Complex II: FAD, FeS

3. Complex III: hemes, FeS

4. Cytochrome C : Heme

5. Complex IV: hemes , Cu, Fe

Complex I :

1. What is it’s other name and what type of proteins is it

2. what is special about it

3. Structure (generally and specifically)

4. Main function specifically

5. what is the function of each part (generally)

6. what is the passage of Electrons in it

7. what opens the proton channels and where

8. How many subunits does it have

1. NADH dehydrogenase Complex (flavoprotein)

2. it is the largest protein component in the inner membrane

3. L shaped structure with:

   1. Hydrophilic Peripheral arm with binding sites of :

      1. FMN/FMNH2

      2. 6 FeS Clusters

      3. UQ

   2. Membrane arm

      1. Composed of transmembrane helices that has 4 proton translocating channels

      2. 1 FeS Cluster

4. Main function: to transfer electrons from NADH to UQ

5. Function :

   1. membrane arm Has 4 proton translocating channels to pump protons to EMM

   2. Peripheral arm accepts Electrons from NADH and H+

   3. UQ shuttles electrons between ETC complexes along inner membrane

6. Just remember the picture honestly

7. the proton channels are opened by a conformational change caused by two stuff

   1. Change in pKa caused by reduction of the membrane arm

   2. a mini-electric current that forms when the electrons are passing

8. 45 subunits

Complex II (Succinate dehydrogenase)

1. What is it also called

2. location

3. What is it’s main function

4. What are it’s subunits

5. how does it differ from other complexes

6. Passage of electrons

1. Also called succinate Ubiquinol reductase

2. Matrix side of the inner mitochondrial membrane

3. To transfer electrons from succinate to Ubiquinone

4. Consists of 4 Subunits :

   1. Subunit ShdA

   2. Subunit ShdB:

   3. Subunit ShdC

   4. Subunit ShdD

5. It doesn’t pump protons into mitochondrial matrix

6. Passage:

   1. ShdA: Fad is reduced to FADH2 using electros from succinate

   2. ShdB: one electron at a time is given to the 3 iron sulfur clusters at it

   3. ShdCD: the 2 electrons are given to UQ to form UQH2 that leaves the complex

1. Subunit ShdA:

   1. What type of a protein is it

   2. Where is it’s location

   3. What does it have

2. Subunit ShdB:

   1. What type of protein is it

   2. What does it have

   3. Where is it’s location

3. Subunits ShdC and ShdD:

   1. What type of proteins are they

   2. What do they have and what is their function

   3. What does have and what is that’s function

1. Subunit ShdA:

   • ShdA is a flavoprotein

   • It extends into the matrix.

   • Contains

     • the succinate binding site

     • covalently bound FAD.

2. Subunit ShdB:

   • iron–sulfur protein

   • three iron–sulfur clusters.

   • It extends into the matrix.

3. Subunits ShdC and ShdD:

   • ShdC and ShdD are integral membrane protein (doesn’t go to the other side)

   • Has:

     • Hydrophobic UQ binding site in between them

       • Gives the electrons from the iron sulfur clusters to UQ

     • A binding site for a heme group

       • Suppresses electron leakage from the complex, preventing oxygen radical formation.

1. What also contributes to Electron transport chain with electrons but isnt considered universal

   1. Location

   2. Function

   3. Mechanism

2. what is similar between them

In some cerain cells there are two flavoprotein enzymes :

1. Glycerol-3-phosphate dehydrogenase:

   • Location: Outer face of the inner mitochondrial membrane (IMM). (interspace)

   • Function: Transfers electrons from cytoplasmic NADH to the UQ in the ETC.

   • Mechanism: Mentioned down

2. Acyl-CoA dehydrogenase + ETF:QO( electron transfer flavoproteins Ubiquinone reductase):

   • Location: Matrix side of the inner mitochondrial membrane (IMM).

   • Function: Transfers electrons to ubiquinone (UQ) from fatty acid oxidation.

   • Mechanism: Acyl-coa gives electrons to FADH2 in Acyl-CoA DH it which gives the electrons to ETF that give electrons to ETF-QO that give electrons to UQ

3. They are both flavoproteins

Complex III:

1. What is it’s other name

2. structure

3. what are cytochromes

4. how do they work and what is the difference between the states

5. what is the main function of the complex

6. what is the formula of the transfer

7. what is the passage of the molecules refered to here

8. electron transfer briefly

9. Where is it’s product released and What is it’s function

1. Cytochrome bc1 complex

2. It is a homodimer with each monomer containing 11 subunits and among them are

   1. cytochromes :

      1. cyt bL

      2. cyt bH,

      3. cyt C1

   2. one Fe-S cluster which mediates UQH2 and Cyt C1

3. Cytochromes are a series of electron transport proteins that contain a heme prosthetic group

4. the heme group’s Iron is reversibly oxidized to carry 1 electron at a time from one to another

   1. Fe2+ is reduced

   2. Fe3+ is Oxidized

5. There are two functions :

   1. Main : to transfer electrons from reduced coenzyme Q (UQH2) to protein called Cytochrome C (Cyt C) not the same as Cyt C1

   2. To pump 4 H+ each time to inter membrane space

6. The general formula is :

   1. UQH2 + 2 Cyt Cox(Fe3+) + 2 H+ Matrix → UBQ + 2 Cyt Cred(Fe2+) + 4 H+ IMS

7. The Q cycle

8. Focus on the down side of the picture

9. Cyt C is released to the intermembrane space carrying one electron at a time to Complex 4

Complex IV:

1. What is it also called

2. what is it’s main function

   1. How many electrons per time

   2. How is this bad

3. Structure

4. Movement of electrons

5. General equation

6. what makes it unique

7. Regulation

1. Cytochrome oxidase

2. catalyzes the four-electron reduction of O2 to form H2O

   1. O2 accept one electron at a time

   2. Might form ROS

3. Homodimer:

   1. Each monomer of the complex contains divided on :

      • 14 cytochromes a and a3.

      • Three copper ions.

      1. Subunit I:

         1. Located centrally

         2. Contains :

            1. Heme a

            2. Binuclear Fe-Cu center: Fromed by

               1. The heme a3

               2. CuB (Copper)

      2. Subunit II:

         1. Contains :

            1. Binuclear Cu-Cu Center: which is 2 CuA molecules (2 Copper)

      3. Subunit III:

         1. facilitates the transport of four protons from the matrix to the IMS

      4. There are more overall (14 subunits)

4. Movement of electrons :

   1. 4 electrons move from Cyt C to CuA in subunit II one at a time

   2. the electrons then move to Cyt A

   3. Then a3-CuB

   4. the reaction starts occuring to

      1. Pump 4 H+ to intermembrane space

      2. Take 4 H+ from matrix one at a time to react them with O2 to finally reduce them and form 2H2O molecules

5. The general equation is :

   1. 4 Cyt C (Fe2+) + 8 H+(matrix) + O2 → 4 Cyt C (Fe 3+) + 2 H2O + 4 H+ ims

6. It is the only complex where electrons don’t leak

7. ATP-binding sites at Cyt c and complex IV which inhibits them and decreases their activity greatly

1. What do those transform to :

   1. NADH

   2. FADH2

2. Why are they different ?

3. write down the general equation of glucose oxidation

1. The two molecules are converted to

   1. NADH = 2.5 ATP (10) = 25

   2. FADH2= 1.5 ATP (2) = 3

2. Because FADH2 only pumps protons at Cmplx 3 and 4 while NADH2 pumps it at 1 3 and 4

3. Glucose + 30 ADP + 30 Pi +6 O2 → 6 CO2 +6 H2O + 30 ATP

1. What are the models that describe the electron transport in the ETC

2. which is the one that is more recent and more supported and why

1. Fluid model:

   1. It describes them as random collisions

2. Solid state model :

   1. States that complexes I, III, and IV are part of a supercomplex called Respirasome (Cmplx II excluded)

   2. States that diffusion is efficient because of the short distances

3. The solid state model is more recent and more supported because the IMM is protein dense (75:25 protein to lipid ratio) which means that random movement is not proper in such folded environment as Protein = Specific

1. What are the types of those that slow or stop or disrupt etc:

   1. Type 1 :

      1. examples mentioned and what do they disrupt

      2. what comes as a result of them

      3. why are they important

   2. Type 2:

      1. What do they do

      2. Examples

   3. Type 3 :

      1. What do they do

      2. Example

   why are type 2 and 3 important

1. ETC: Inhibitors

   1. 3 mentioned :

      1. Antimycin A: inhibits Cyt b in Complex III

      2. Complex I is inhibited by :

         1. Amytal

         2. Rotenone:

      3. complex IV is inhibited by:

         1. Carbon monoxide(CO)

         2. Azide(N3-)

         3. Cyanide (CN-)

   2. Results:

      1. Everything that is before them is in reduced form

      2. Everything after them is in oxidized form

      3. O2 consumption is reduced or stopped

   3. It has two significant functions:

      1. It allows us to determine the correct order of electron movement by measuring O2 consumption

      2. It kills cancer in chemotherapy

2. ETC-uncouplers:

   1. They balance out the electrochemical gradient by equalizing the proton concentrations

   2. Examples include :

      1. Dinitrophenol diffuse through the membrane picking up protons from one side and passing with them to the other side to release them there

3. Ionophores :

   1. Hydrophobic molecules that dissipates osmotic gradients by inserting themselves into the membrane and forming a channel allowing for the passage of ions

   2. Example includes gramicidin

type 2 and 3 are important because they dissipate energy in the form of heat

1. List the uncouplers

1. UCP1 (thermogenin) :

   1. Only in mitochondria of brown fat

      1. 10 % of protein in mitochondrial inner membrane in them

   2. Activated by fatty acids

   3. Causes Non-shivering thermogenesis (provides heat)

2. UCP2 : Used to control ROS formation

3. UCP3:

   1. Skeletal muscle and brown adipose tissue

   2. roles in fatty acid oxidation and decreasing ROS

4. UCP4 and UCP5 found in CNS

1. what theory explains how energy is generated from ETC

   1. what does it say

   2. what does electrochemical mean and difference in and out

   3. What ensures that this energy is created

2. What supports the theory

1. The chemiosmotic theory:

   1. Explains that the proton (H+) electrochemical gradient created by the ETC contains energy harvested from the NADH and FADH2 and energy can be taken from the electrochemical gradient by two ways by moving from Intermembrane space to matrix side again:

      1. Passage through ATP synthase to synthesize ATP and store energy in form of Pi bond to ADP

      2. Passage through the membrane caused by uncouplers and ionophores which dissipates energy in form of heat

      3. performing work in other forms

   2. Δp : Electrochemical gradient also called the protonmotive force :

      1. Ψ: Electrical gradient caused by different charges on other sides more positive in intermembrane space due to more protons measured in 150 mV charge difference (negative in matrix)

      2. ΔpH : Chemical gradient formed by more protons concentration outside measured in 0.5 pH units difference (more negative outside )

   3. The inner membrane is not permeable to Ions like H+ so they have to pass through special channels

2. Evidence include:

   1. pH drops when O2 is added to intermembrane space and pH difference 8 inside and 7.5 outside

   2. ATP synthesis stops when inner membrane is disrupted which allows for passage of protons without harvesting their energy

   3. Uncouplers and ionophores stop ATP synthesis by disrupting the flow of H+

Complex V :

1. What it’s other name

2. What are the unit + What is one another name + What are the ratios of the subunits it has

   1. What is it divided to

      1. What does that division contain

         1. What forms it

         2. What is the function of it

3. step by step work

4. what inhibits it

1. ATP synthase

2. Structure consists of two major components :

   1. F1 Unit Also called Active ATPase (α3:β3:γ:δ:ε) :

      1. flexible stator:

         1. α,β hexamer

            1. α3 along with β3 form the thing that looks like a mushroom cap

            2. Has 3 nucleotide binding sites for ATP synthesis

         2. δ Subunit:

            1. Connects the α,β hexamer to the bb of F0 superiorly

      2. Rotor:

         1. Central Shaft:

            1. γ and ε form it

            2. connects F1 to F0 inferiorly

   2. F0 Unit Also Proton Channel (a:b2:c10–12.):

      1. The same flexible stator as above:

         1. b2 units:

            1. Connect F0 to F1

         2. A unit:

            1. Has a channel through which H+ can pass forcing the rotor to rotate

      2. Rotor No 2 :

         1. C ring:

            1. Formed by C10 to 12 units

            2. Rotates by A unit and delivers this rotation to rotor 1 of the F1 unit

3. Step by step :

   1. Proton enters c ring through (a) subunit

   2. this causes C ring to rotate counterclockwise by protonmotive force

   3. which causes ε and γ subunits of F1 to rotate

   4. α,β hexamer is being pushed to rotate by rotor 1 but is prevented by the stator

   5. each proton that enters causes the α,β hexamer to rotate by 120°

   6. this causes a conformational change on all 3 nucleotide binding sites which allows for ATP synthesis over 3 sequential rotations by

      1. ADP and Pi bind to an L site

      2. ATP is synthesized when the L conformation is transformed to a T conformation

      3. ATP is released as the T conformation converts to an O conformation

   7. Quick note ATP cannot be released from the O site unless ADP and Pi are bound to the adjacent T conformation

4. inhibited by Oligomycin which binds to subunit a to block entery

1. Where are the nucleotide binding sites located

2. What are they

   1. What Occurs in each

1. In F1 Unit α,β hexamer :

   1. L : Inactive (binds to ADP and Pi)

   2. T : active with high affinity to ATP

   3. O: inactive releases ATP

1. How can we measure coupling

2. what is the maximum in different molecules

3. At which rate does it usually occur

1. by the P:O ratio which is (the number of moles of Pi consumed for each oxygen atom reduced to H2O)

2. the maximum ratio for oxidation is :

   1. NADH: 2.5

   2. FADH2 : 1.5

3. At the maximum rate

1. How is the Oxidative phosphorylation controlled

1. By respiratory control : (ratio of ATP/ADP Pi)

   1. more ADP is more O2 reduced

   2. More ATP is inhibition of the ATP synthase so less O2 reduced

2. By controlling respiratory control :

   1. The amounts of ATP and ADP in the mitochondria are controlled by two transport proteins :

      1. The ADP-ATP translocator also Adenine nucleotide translocator :

         1. Dimeric protein

         2. Works by 1:1 exchange of Cytoplasmic ADP for Mitochondrial ATP which is favored because (more - inside due to ATP)

         3. Increases ADP in thus more ATP synthesis

      2. Phosphate translocase also H2PO4-/H+ Symporter

         1. Carries the phosphate ion from the cytoplasm towards the matrix along with the H+ ion

         2. Increases Pi in thus more ATP synthesis

How many H+ are used and for what

1. A total of 4:

   1. Three H+ are used for rotation of ATP synthase rotor and formation of ATP

   2. one H+ is used to carry the Pi from the cytoplasm towards the matrix

1. What are the problems with the use of ATP

2. what solves it

3. how

1. There are two problems :

   1. ATP diffuses slowly through the interior of the cell which fail to reach it’s demanded place

   2. ADP and Pi and H+ must be quickly removed to avoid inhibition of ATPases

2. It is solved by creatine kinase/phosphocreatine shuttle system by

   1. Phosphocreatine has lower molecular mass so it diffuses quickly within cell

Write down the reaction related to above

PCr2- + Mg ADP- + H+ → Cr+ Mg ATP2-

What are the isozymes of Creatine Kinase

1. 2 Cytoplasmic:

   1. Composed of two subunits which can be : either B (brain) or M (muscle)

      1. Homodimer :

         1. MMCK : occurs in muscles and heart

         2. BBCK : occurs in brain and heart

      2. Hetero:

         1. MBCK : occurs in heart

2. Mitochondrial :

   1. Octomers

   2. Located in intermembrane space

   3. bound to cardiolipin (imm phospholipid) and ANT and VDAC

      1. ANT : ATP from Matrix to IMS

      2. VDAC : From IMS to cytoplasm

   4. Two types :

      1. Ubiquitous MtCK: found in non-muscle cells

      2. Sarcomeric mtCK:

What uses the most ATP

1. NaK atpase

2. Ca2+ ATpase

Complete oxidization of Glucose go on write the table

WOW keep in mind that the NADH and FADH2 are converted to ATP by the amount of H+ that they draw to the Inetermembrane space and as FADH2 comes at a later stage of ETC it transports less H+ to intermembrane space and thus generating less ATP

What are the shuttles to transport what is the difference between them

1. Glycorl-3-phosphate shuttle

2. Malate asparatate shuttle

   1. More Efficient

   2. more complex

1. Glycerol-3-phosphate shuttle

   1. Draw it

1. Malate-Aspartate shuttle

2. Draw the picture

glutamate + OAA → Transaminase → A-ketoglutarate + aspartate